AU2006207365B2

AU2006207365B2 - Formulation

Info

Publication number: AU2006207365B2
Application number: AU2006207365A
Authority: AU
Inventors: Allan Kunamoney Nadian
Original assignee: Food And Environment Research Agency Fera Representing Secretary Of State For Environment Fo
Current assignee: UK Secretary of State for Environment Food and Rural Affairs
Priority date: 2005-01-19
Filing date: 2006-01-19
Publication date: 2011-10-13
Anticipated expiration: 2026-01-19
Also published as: US20080076666A1; WO2006077394A3; EP1838148A2; AU2006207365A1; GB0501030D0; CA2594628C; CA2594628A1; WO2006077394A2

Abstract

A formulation comprising an active component, a microcapsule and a dye, which protects the active component from degradation by U. V light. Such formulations can be used in a variety of applications including agrochemical applications, which are also described and claimed.

Description

WO 2006/077394 PCT/GB2006/000157 1 Formulation The present invention relates to formulations comprising an 5 active component, such as an agrochemical, a pharmaceutical, a cosmetic or a veterinary compound, a microcapsule and a dye, as well as to components used in the formulations, and to methods of using them. 10 Microcapsules have been found to be a very effective tool for aiding the delivery of active components such as chemical and biological substances to a target environment. In particular they have been found to be useful delivery vehicles for chemicals and biological substances. For instance, they can be 15 manufactured to release their contents only under suitable conditions of pH, temperature or moisture etc. Problems may occur however on storage or in use, due to degradation of the active component as a result of exposure to 20 U.V. radiation. These problems occur particularly where the active component is U.V. labile, as many pharmaceutical and agrochemical substances are. In the case of agrochemicals, the problem may be aggravated by the fact that in use, the compounds may be exposed to high levels of U.V. radiation. 25 U.V. protectants such as benzophenones: 2-hydroxy-4-n octoxybenzophenone and 2,2'-dihyroxy-4,4' dimethoxybenzophenone; benzotriazoles: 2-(2-hydroxy-5' methylphenyl)-benzotriazole and 2-(3',5'-diallyl-2' hydroxyphenyl)benzotriazole; and free radical scavengers: 30 bis(2,2,6,6-tetramethyl-4-piperidyl)sebecate and 8-acetyl-3 dodecyl-7,7,9,9-tetramethyl-1,3,8-triazaspiro(4.5)decane-2,5 dione are known, but may not be sufficient to provide adequate protection for the compounds under these circumstances. 35 Other issues may arise in relation to the detection of formulations once applied to a target. For instance, in the WO 2006/077394 PCT/GB2006/000157 2 case of a topically applied medication, or agrochemical applied by spraying techniques, it may be difficult to see whether adequate or complete coverage has been achieved. 5 The present invention provides an improved microcapsule formulation. The applicants have prepared a formulation comprising an active component, for example an agrochemical, pharmaceutical or 10 cosmetic, a microcapsule and a dye. According to a first aspect of the present invention there is provided the use of a dye for the protection of an active component contained within a microcapsule from U.V. 15 degradation. The presence of the dye, in particular one that absorbs U.V. light may protect the active component from U.V degradation. Thus the presence of the U.V absorbing dye would be expected to 20 increase the half life of the active component on exposure of the formulation to U.V. light. Additionally or alternatively, it may also provide a means for detecting the formulation after application. 25 The dye is preferably incorporated within or located on the surface of the microcapsule but may instead be free from the microcapsules, for example in a solution surrounding the microcapsules. 30 As used herein, the term "dye" refers to any material which can be detected visually, and/or which absorbs UV radiation. Suitably it is able to colour or stain material it comes into contact with. Ideally the dye is dissolved in a suitable solvent. A solution of the dye may advantageously allow for 35 homogeneous dying of the microcapsule.

WO 2006/077394 PCT/GB2006/000157 3 The dye is suitably one that allows visible monitoring of the application of such formulations to, for example, the surface of a plant, or the skin of a human or animal. Applying a formulation, which contains an encapsulated active component, 5 for example an agrochemical and a dye, would give a visual indication as to which plants have been treated and which plants have not been treated, therefore, ensuring that none are missed or repeated by accident. 10 Formulations of this type, for example, a pesticide formulation, a sun tan lotion formulation or a topical medicine formulation, when applied, would leave a mark on the skin of the animal such as human to whom it is applied, giving a visual indication of the areas of skin to which the formulation has 15 and has not been applied. Most preferably the dye is an environmentally acceptable dye. In general, this will mean any dye that is permitted in food, drug, cosmetic and pesticide formulations by the relevant 20 government bodies. Thus such dyes are either agriculturally, pharmaceutically or veternarily acceptable dyes. Preferably the dye is Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, 25 Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown, or combinations thereof. However, a silver stain may be employed when this is not 30 incompatible with the end use of the formulation. In a particular embodiment, the dye is any dye that has a U.V. absorption spectrum similar to that of Bismark Brown. By "similar" it is meant that the peak absorption occurs at 35 approximately the same wavelength as the peaks of the Bismark Brown spectrum, and/or is a dye listed in Table 1 below.

WO 2006/077394 PCT/GB2006/000157 4 Most preferably the dye is Chocolate Brown. (Brown 3, CI 20285, E155, WS Simpson, London), which has the following chemical structure: 5 NaO 3 S N=N OH N=N SO 3 Na \ / HO CH20H Chocolate Brown The active component is suitably encapsulated within or located on the surface of the microcapsule but may instead be free from 10 the microcapsules, for example in a solution surrounding the microcapsules. Preferably however, the active component is encapsulated within the microcapsule. The active component may comprise a living or non-living 15 component. Suitable living components are bacteria, nematodes, viruses or fungi, which may or may not be inactivated or attenuated. Preferably the active component is a non-living component, such as a chemical compound, or a reagent that is derived from a living component, for example an immunogen such 20 as a polypeptide or protein, as well as killed microorganisms such as heat or chemically killed bacteria and/or viruses The active components are suitably agrochemical, pharmaceutical, cosmetic or veterinary reagents. 25 Suitable cosmetic reagents include perfumes and other fragrances. In a particular embodiment the active ingredient is other than 30 an anti-bacterial component.

5 Most preferably the microcapsules comprise an agrochemical which herein shall be taken to include pesticides such as insecticides, acaricides, fungicides and herbicides, as well as plant growth regulators and fertilizers. Such formulations would be very useful in the field of agriculture and horticulture where spraying with such agrochemicals is very common. Most preferably the agrochemical is a pesticide for example, a fungicide and especially an insecticide or acaricide. The agrochemical may be UV labile, in the sense that it is unstable or degrades over time, when exposed to U.V. light. Inclusion of certain dyes, in particular chocolate brown, may, in some circumstances reduce any phytotoxic effect of such chemicals on the plants to which they are applied. The use of these dyes as safeners therefore forms a further aspect of the invention. Suitable agrochemicals are naphthoquinone derivatives. The term "naphthoquinone derivative" shall be taken herein to mean any agriculturally useful compound containing a naphthalene core, substituted by two oxo groups, and suitably one or more further substitutents. In particular, they will comprise 1,2-naphthoquinone or 1,4-naphthoquinones which carry one or more further substitutents. The naphthoquinone derivative may be a synthetic compound or it may be derived from a natural source. For instance, the active component may comprise an isolated extract from a species of Calceolaria plant for example Calceolaria sessilis, Calceolaria andina or Calceolaria glabrata var. meyenenis which are known to contain naphthoquinone derivatives. Examples of suitable compounds are described for instance in WO 97/16970, WO 95/32176, US4970328, US4929642, W096/21355, WO 2006/077394 PCT/GB2006/000157 6 W096/21354, W097/02271 and EP1051909 and these are incorporated herein by way of reference. Suitable further substituents as defined above include, for 5 instance, hydroxy, alkoxy, aryloxy, aralkyloxy, alkanoyloxy, alkylsulphonyloxy, arylsulphonyloxy, alkyl, alkenyl, halogen, nitro, cyano, amino, mono- or di-alkylamino, alkoxycarbonyl, carboxyl, alkanoyl, alkylthio, alkylsulphinyl, alkylsulphonyl, carbamoyl, alkylamido, cycloalkyl, aryl, aralkyl; wherein any 10 alkyl, alkenyl or aryl groups or moieties within the groups may be optionally substituted by one or more halo, trifluoromethyl, trifluoromethoxy, trifluoromethylsulphenyl, trifluoromethylsulphonyl, trimethylsilyl, or cyclohexyl which is optionally substituted by methyl, trifluoromethyl or 15 trimethylsilyl. Alternatively, substituents on adjacent positions on a naphthoquinone ring can be joined together to form an optionally substituted ring which may be saturated or 20 unsaturated, and may contain one or more heteroatoms selected from oxygen, sulphur and nitrogen. The ring suitably comprises from 3 to 7 atoms, for instance, 5 atoms, and in particular is a fused tetrahydrofuran ring. Suitable substitutents for a ring formed in this way may include one or more alkyl groups 25 such as methyl. A particular example of such a compound is dunnione, as described in WO 97/16970. As used herein, the term "alkyl" refers to straight or branched chains containing from 1 to 20, suitably from 1 to 13 carbon 30 atoms. The term "alkenyl" refers to straight or branched chains of from 2 to 20, suitably from 2-13 carbon atoms. The term "aryl" refers to aromatic groups such as phenyl or naphthyl, and "aralkyl" refers to alkyl groups carrying an aryl substituent such as benzyl. The term "halo" includes chloro, 35 bromo or fluoro.

7 Particular naphthoquinone derivatives are l,4-naphthoquinone derivatives of general formula (I) 0 R 0 (I) where R' is selected from an optionally substituted alkyl group, a hydroxy group or a group -OCOR 4 where R 4 is selected from hydrogen,

C

1

-

12 alkyl, Ci- 12 haloalkyl, C 1

-

12 hydroxyalkyl, C 1

-

1 2 carboxyalkyl, phenyl or benzyl. In particular, R is suitably selected from hydroxy of a group OCOR 4 . Preferred groups R 4 are hydrogen, C 1

.

6 alkyl, C 1

-

6 haloalkyl, phenyl or benzyl.

R

2 is, in particular, is an alkyl, or alkenyl group as defined above, which may be optionally substituted, in particular with a group silicon containing group such as -Si(RsR 6 R7) where R 5 , R 6 and R 7 each represent a C 1

-

4 alkyl group, such as methyl. Particular preferred naphthoquinone derivatives are compounds of formula (III), (IV) or (V) as set out below (and as described in Pest Management Science, 2001, 57 (8) p749-50) , or a combination of such compounds.

WO 2006/077394 PCT/GB2006/000157 8 Compound No. III O CH3 0

CH

2 O H3C CH 3 IV 0 CH V0 /Si-CH3 O~ CH Si-CH3 O CH3 H30 CH3 Most preferably the naphthoquinone derivative is compound (V) shown above. 5 Naphthoquinone derivatives such as those described above have been found to be very effective at killing pests, for example Bemisia tabaci (tomato plant pest), Psoroples cuniculi (rabbit ear canker mite), Dermanyssus gallinae (poultry red mite), 10 Psoroples ovis (Sheep scab mite), Musca domestica (housefly) and Blatella germanica (German cockroach). These chemicals are however photo labile to varying degrees and therefore in there natural state, degrade in UV light. The use 15 of conventional UV protectants either alone or in combination with free radical scavengers (such as bis(2,2,6,6-tetramethyl- WO 2006/077394 PCT/GB2006/000157 9 4-piperidyl)sebecate and 8-acetyl-3-dodecyl-7,7,9,9 tetramethyl-1,3,8-triazaspiro(4.5)decane-2,5-dione) and/or antioxidants (such as dibutylhydroxy toluene [BHT]) failed to prevent photodegradation of these compounds. 5 Formulations according to the present invention comprise a dye which preferably can absorb UV light and therefore allow agrochemicals such as those described above to be delivered using microcapsules where previously microcapsule delivery of 10 U.V. labile compounds would not have been effective. The microcapsules can be formed from any suitable substance, for example gelatine, polyurethane, polyamide, polyurea, polyester or a biodegradable polymer for example Poly-lactide 15 (PLA), but most preferably are comprised of gelatine or polyurethane. They may be prepared using any conventional method, such as the complex coacervation method or the interfacial polymerisation 20 method. These methods are suitably carried out in the presence of the active component so that this becomes encapsulated within the microcapsules. The encapsulation may also be carried out in the presence of the dye, so that this may also be incorporated into the microcapsules, either encapsulated 25 within them, or in the surface layer. Alternatively or additionally, dye may be applied subsequently to the prepared microcapsules. 30 The microcapsules suitably have an average diameter of less than 60pm, but preferably an average diameter of 50pm and most preferably are between 3 and 35 pm in diameter. In a preferred embodiment, the microcapsule has particulate 35 matter located in a wall thereof to render the wall permeable.

WO 2006/077394 PCT/GB2006/000157 10 This can be achieved by preparing the microcapsules in the presence of the particulate matter. As used herein, the term "particulate matter" includes any 5 small solid particles, including microparticles such as microspheres, and nano particles (whose dimensions are less than lpn). In particular, in this embodiment, the microcapsules are 10 comprised of a material, which is generally impermeable under most conditions such that the active component may be contained within the microcapsule. However, the presence of a particulate matter such as a nano particle or microsphere located in a wall thereof renders the wall permeable. 15 This permeability may be caused in various ways, for example a nano particle or microsphere may act as a wick allowing the active component to move out of the microcapsule by capillary action, or may instead or additionally allow the active 20 component to move out of the microcapsule by some other action. Microcapsules of this type advantageously allow for the controlled delivery of a substance encapsulated within the microsphere and are particularly useful where a slow release is 25 required. The particulate matter can be of any suitable material, which is compatible with the other components in particular the wall, of the microcapsule. They are suitably insoluble in conditions 30 in which the microcapsule is to be stored or used. The particles of the particulate matter are suitably of sufficient size to ensure that when positioned in the wall of the microcapsule, the microcapsule is rendered permeable. Since 35 the properties of the wall may vary, the size and type of the particles will need to be selected to be compatible with the WO 2006/077394 PCT/GB2006/000157 11 type of microcapsule being used. Most suitably the particles of the particulate matter are of a size, which ensures that the particles traverse the wall of the microcapsule. Suitably the particles are less than 30pm, preferably between 0.10 and 20pm, 5 more preferably between 0.10 and 10pm and most preferably have an average diameter of 0.40 micrometers. In a particular embodiment, the particulate matter comprises nano particles. Particular examples of particulate matter include inorganic 10 particles such as metals, for instance titanium, iron, copper, silver, gold, lead, tin, aluminium, or insoluble salts therefore, including metal oxides. A particular example of such a particle is titanium dioxide. 15 Alternatively, the particulate matter may be of an insoluble polymeric material. Suitable materials include insoluble polymers such as insoluble polysaccharides, polyacrylates, polymethacrylates, polyacrylic acids, polymethacrylic acids, polyalkylenes such as polythenes, polyurethanes or 20 polystyrenes, or copolymers of these. Particularly suitable polymers include polysaccharides such as cellulose or derivatives thereof, such as alkyl cellulose, for instance ethyl cellulose. 25 Suitably the particles of the particulate matter are coated with a material that enhances permeability through the microcapsule. A particular example of such a material is silica. In particular, where the particles are inorganic particles as described above, a silica coating has been found 30 to be particularly useful in enhancing the permeability inducing properties of the particles. This may be due to some wicking effects. Thus, in a particular embodiment, the particulate matter comprises silica coated titanium dioxide nano particles, such as the material available commercially as 35 Ti-Puree and in particular Ti-Pure® R-931.

WO 2006/077394 PCT/GB2006/000157 12 The presence of particulate matter, such as nano particles, for example titanium dioxide particles, and in particular silica coated titanium dioxide particles, such as Ti-Pure® R-931 may 5 have an additional advantage of inhibiting aggregation of the dispersed droplets during production of the microcapsules. Sometimes during the preparation of the microcapsules, especially capsules below 50 microns, the dispersed droplets are encapsulated as aggregates resulting in bigger capsules. 10 The presence of particulate matter may inhibit this aggregation enabling discrete small microcapsules to be formed. These smaller microcapsules may be preferred as they can be easier to apply to a plant or an animal by spraying, as they do not clog up the nozzle of any spraying device. 15 The permeability of the microcapsule may further be enhanced by combining the particulate matter with a leachable material, which is subsequently, at least partially leached out of the resultant microcapsule wall. This appears to enhance the 20 permeability of the resultant microcapsule in certain circumstances. Particular examples of suitable leachable materials include certain polymers, for instance copolymers of methacrylates and 25 methacrylic acid. A particular example is a copolymer of .cationic dimethylaminoethylmethylmethacrylate and neutral methacrylic acid ester, for instance as available commercially as Eudragit E100 (Degussa, Dusseldorf). 30 Eudragit E100 is a copolymer of cationic dimethylaminoethylmethyl methacrylate and neutral methacrylic acid ester having the following structure:- WO 2006/077394 PCT/GB2006/000157 13

CH

3

CH

3 ...- CH 2 - C-CH 2 - C-... O OR

CH

2

/CH

3

CH

2 -N

\CH

3 R = CH 3 , C 4

H

9 This is suitably incorporated into the wall of the impermeable 5 microcapsules as described above. It can be leached using hydrochloric acid, in particular 1M HCl using conventional conditions. Typically the microcapsules were suspended in aqueous 1M HCl with agitation at room temperature for 18 hours to leach the Eudragit E100 from the capsule wall. The capsules 10 were subsequently washed thoroughly and resuspended in water. In a preferred embodiment a silica coated particle, for example, a titanium dioxide particle such as Ti-Pure@ R-931 is located in a wall of the microcapsule to render said wall 15 permeable. Titanium dioxide is however at least partially phytotoxic to some plants, and therefore the use of a dye which is capable of reducing the phytotoxic effects of -the titanium dioxide is 20 preferable. Chocolate Brown is particularly efficient at reducing the phytotoxic effects of titanium dioxide and is therefore preferred for use in conjunction with titanium dioxide particles. 25 The formulation suitably further 'Comprises a suitable carrier or excipient. These may be solid or liquid excipients and will be selected in accordance with routine practice in the particular field. For instance, agrochemical formulations will WO 2006/077394 PCT/GB2006/000157 14 generally further comprise an agriculturally acceptable carrier or diluent as is known in the art. Concentrates in the form of solids or liquids may be prepared, which require dilution in water prior to application, for example by spraying. 5 The formulation can be formed into, for example, water dispersible granules, slow or fast release granules, soluble concentrates, oil miscible liquids, ultra low volume liquids, emulsifiable concentrates, dispersible concentrates, oil in 10 water, and water in oil emulsions, micro-emulsions, suspension concentrates, aerosols, capsule suspensions and seed treatment formulations. The formulation type chosen in any instance will depend upon 15 the particular purpose envisaged and the physical, chemical and biological properties of the formulation. Granules may be formed either by granulating a formulation as described above and one or more powdered solid diluents or 20 carriers. One or more other additives may also be included in granules, for example an emulsifying agent, wetting agent or dispersing agent. Dispersible concentrates may be prepared by mixing a 25 formulation as described above in water or an organic solvent, such as a ketone, alcohol or glycol ether. These dispersions may contain a surface-active agent. Suspension concentrates may comprise aqueous or non-aqueous 30 suspensions of formulations as described above. Suspension concentrates may be prepared by combining the formulation in a suitable medium, optionally with one or more dispersing agents, to produce a suspension of the microcapsules. One or more wetting agents may be included in the suspension and a 35 suspending agent may be included to reduce the rate at which the microcapsules settle.

WO 2006/077394 PCT/GB2006/000157 15 Aerosol versions of the formulations may further comprise a suitable propellant, for example n-butane. A formulation as described above may also be dispersed in a suitable medium, for 5 example water or a water miscible liquid, such as n-propanol, to provide formulations for use in non-pressurised, hand actuated spray pumps. Agrochemical formulations may further include one or more 10 additives to improve the biological performance, for example by improving wetting, retention or distribution on surfaces; resistance to rain on treated surfaces; or uptake or mobility of the formulation. Such additives include surface active agents, spray additives based on oils, for example certain 15 mineral oils or natural plant oils (such as soy bean and rape seed oil), and blends of these with other bio-enhancing adjuvants. Formulations as described above may also be adapted for use as 20 a seed treatment. Wetting agents, dispersing agents and emulsifying agents may be surfactants of the cationic, anionic, amphoteric or non-ionic type, as is known in the art. 25 Suitable suspending agents which may be included in the formulations include hydrophilic colloids (such as polysaccharides, polyvinylpyrrolidone or sodium carboxymethylcellulose) and swelling clays (such as bentonite 30 or attapulgite). The formulations may also contain other compounds having biological activity, for example micronutrients or other agrochemicals having similar or complementary activity. 35 WO 2006/077394 PCT/GB2006/000157 16 Pharmaceutical compositions comprising formulations of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups 5 or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral 10 administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular dosing or as a suppository for rectal or vaginal dosing. The pharmaceutical compositions may be obtained by conventional 15 procedures using conventional pharmaceutical excipients, well known in the art. Aqueous suspensions generally contain the microcapsules together with one or more suspending agents, dispersing or 20 wetting agents. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame). 25 Oily suspensions may be formulated by suspending the microcapsules in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a 30 thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These pharmaceutical compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. 35 WO 2006/077394 PCT/GB2006/000157 17 Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by mixing a formulation as described above with a conventional, topically acceptable, vehicle or diluent using 5 conventional procedure well known in the art. For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), 10 Pergamon Press 1990. The amount of active component that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the 15 particular route of administration. In particular, the formulation comprises an agrochemical. According to another aspect of the present invention there is 20 provided a microcapsule which contains an agrochemical. The microcapsule preferably further comprises a dye. Suitable agrochemicals are as described above, but in particular are pesticides, such as insecticides, and suitably 25 those which are photo labile. The microcapsule preferably encapsulates the agrochemical, but alternatively the agrochemical may be dispersed throughout the microcapsule or be present only on the surface thereof. 30 The dye preferably coats the surface of the microcapsule but may instead or additionally be dispersed throughout the microcapsule. In a preferred embodiment the microcapsule is formed from gelatine, encapsulates Compound (V), is dyed with Chocolate Brown and has silica coated titanium dioxide 35 particles such as Ti-Pure@R-931 particles dispersed in the wall thereof.

18 Generally agrochemical formulations will be delivered using conventional large scale spray equipment. However, for certain horticultural or pharmaceutical applications, formulations may be incorporated into suitable delivery devices such as atomisers, nebulizors or spray guns. According to another aspect of the present invention there is provided a formulation delivery device, such as an atomiser, nebulizor or spray gun containing a formulation as described above. The atomiser, nebulizor or spray gun can be used to apply the formulation to its intended target. For example if the microcapsules contain a pesticide or insecticide the atomiser, nebulizor or spray gun can be used to apply the formulation to a plant, animal or its environment to provide protection from pests. The formulations may be in the form of a dispersion of a solid in a gas or liquid. These may be prepared for example, from suspensions of the formulation in a liquid such as water, using a device such as a nebulizer, or from dry powders. In the case of a nebulized aerosol, the dispersion comprises essentially wet microcapsules in air. According to another aspect there is provided a formulation comprising an agrochemical, a pharmaceutical or a cosmetic, a microcapsule and a dye, preferably wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown. According to a further aspect, there is provided a formulation comprising an active component, a microcapsule and a dye selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown.

18A According to another aspect, there is provided a reagent delivery device containing a formulation according to the present invention or a microcapsule according to the present invention. According to a further aspect, there is provided a use of a dye, wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown, as a safener for the reduction of the phytotoxic effects of reagents on plants. According to another aspect of the invention there is provided a method of protecting a plant, said method comprising administering to the plant or its environment a formulation comprising (i) a microcapsule comprising an active component, and (ii) a dye, wherein components (i) and (ii) can be applied together or in separate stages, and wherein the active component is an agrochemical for example a pesticide such as an insecticide. Preferably the agrochemical is a naphthoquinone derivative and the dye is capable of absorbing UV light. Suitably the dye is included in the microcapsule, and the administration takes place in a single step. The formulation may be applied by any of the known means of applying agrochemical compounds. For example, it may be 19 applied, formulated or unformulated, to the pests or to a locus of the pests (such as a habitat of the pests, or a growing plant liable to infestation by the pests) or to any part of the plant, including the foliage, stems, branches or roots, to the seed before it is planted or to other media in which plants are growing or are to be planted (such as soil surrounding the roots, the soil generally, paddy water or hydroponic culture systems), directly or it may be sprayed on, dusted on, applied by dipping, applied as a cream or paste formulation, applied as a vapour or applied through distribution or incorporation of the formulation in soil or an aqueous environment. Formulations as described above may be sprayed onto vegetation using electrodynamic spraying techniques or other low volume methods, or applied by land or aerial irrigation systems. Formulations as described above may be supplied in the form of a concentrate, the concentrate being added to water before use. These concentrates, are often required to withstand storage for prolonged periods and, after such storage, to be capable of addition to water to form aqueous preparations which remain homogeneous for a sufficient time to enable them to be applied by conventional spray equipment. Such aqueous preparations may contain varying amounts of the formulation (for example 0.0001 to 10%, by weight) depending upon the purpose for which they are to be used. According to another aspect of the invention there is provided a method for producing an effective microcapsule comprising encapsulating an agrochemical, pharmaceutical or cosmetic within the microcapsule, and dyeing the surface of the microcapsule and/or incorporating dye into the microcapsule during its preparation. Preferably the active component is a pharmaceutical or agrochemical. Most preferably the wall of the microcapsule is permeable.

WO 2006/077394 PCT/GB2006/000157 20 Microcapsules produced in this way can be included into agrochemical compositions, for instance by combining them with agriculturally acceptable carriers as described above. 5 The work revealed here has shown that titanium dioxide and particularly silica coated titanium dioxide particles are at least partially phytotoxic to some plants. This may be due to the desiccating effect caused by the silica on the surface of the titanium dioxide particles and the photocatalytic effect of 10 titanium dioxide. This finding opens up the possibility that these particles could be used as herbicides, in particular as broad-spectrum dessicants. Thus according to yet a further aspect of the invention, there 15 is provided a method for killing or controlling plants by application of titanium dioxide particles, and particularly silica coated titanium dioxide particles thereto. These particles will generally be applied in the form of a 20 herbicidal composition, in which they are combined with agriculturally acceptable carriers and such compositions form yet a further aspect of the invention. According to a further aspect of the invention there is 25 provided a method for protecting an active ingredient encapsulated within a microcapsule from U.V. degredation comprising dyeing the surface of the microcapsule and/or incorporating dye into the microcapsule and/or suspending the microcapsule in a dye. The active ingredient may be an. 30 agrochemical, pharmaceutical or cosmetic. The invention will now be particularly described by way of example and with reference to the following Figures. 35 Figure 1, shows the schematic protocol for the first bioassay.

WO 2006/077394 PCT/GB2006/000157 21 Figures 2a and 2b, show photographs of an idealised clip cage arrangement on tomato plant leaves. Figure 3, shows the schematic protocol for the second Bioassay. 5 Figures 4 shows UV absorption spectra of Chocolate Brown and Bismarck Brown R. Figure 5 shows Chocolate Brown irradiated with 254 nm UV light. 10 Figure 6, shows the stability of COMPOUND (V) in undyed and Chocolate Brown (CB) dyed impervious gelatine microcapsules exposed to daylight. 15 Figure 7, shows a calibration curve for quantification of COMPOUND (V) by HPLC. Correlation coefficient(R 2 ) = 0.9996 Figure 8a to 8d, show SEM micrographs of various microcapsules showing their surface morphology. 20 Figure 9a to 9b, show SEM micrographs of Ethylcellulose and Eudragit E100 microspheres embedded in the walls of microcapsules. 25 Figure 10a and 10b, show photomicrographs of capsule distribution pattern obtained with (a) 1/8 and (b) 1/4 dilution of spray solution on filter paper. Figure 10c, shows a photomicrograph of capsule distribution 30 pattern obtained with 1/6 dilution of spray solution on the abaxial surface of tomato leaf. Figure lla, shows mean mortality of B.tabaci in Bioassay 1, exposed to daylight. 35 WO 2006/077394 PCT/GB2006/000157 22 Figure llb, shows mean mortality of B.tabaci in Bioassay 1, exposed to subdued light. Figure lic, shows a comparison of mortality of B.tobaci between 5 daylight and subdued light after 24 hour exposure in bioassay 1. Figure 1ld, shows a comparison of mortality of B. tobaci between daylight and subdued light after 48 hour exposure in bioassay 10 1. Figure lle, shows a comparison of mortality of B.tobaci between daylight and subdued light after 4 days exposure in bioassay 1. 15 Figure llf, shows a comparison of mortality of B. tobaci between daylight and subdued light after 7 days exposure in bioassay 1. Figure 12a, shows mean mortality of B. tabaci in Bioassay 2, exposed to subdued light. 20 Figure 12b, shows mean mortality of B. tabaci after 1 day in Bioassay 2. Figure 12C, shows mean mortality of B.tabaci after 2 days in 25 Bioassay 2. Figure 12d, shows mean mortality of B.tabaci after 4 days in Bioassay 2. 30 Figure 12e, shows mean mortality of B. tabaci after 7 days in Bioassay 2. Figure 13a, shows an SEM micrograph of gelatine microcapsule (mean diameter 50 pm) with Ti-Pure* R-931 incorporated in the 35 wall.

WO 2006/077394 PCT/GB2006/000157 23 Fig. 13b, shows an SEM micrograph of artificially broken gelatine capsule showing the distribution of Ti-Pure* R-931 in the wall. 5 Figure 14, shows a photograph of tomato plants two days after treatment with various Ti-Pure@ R-931 incorporated gelatin microcapsule formulations (A-in middle with label hidden, B, C, D & E) as per Bioassay 2. F-no treatment (absolute control). 10 Figure 15, shows photographs of tomato plants two days after treatment with various R- Ti-Pure @931 incorporated gelatin microcapsule formulations as per Bioassay 2. Treatment B: Ti Pure® R-931 + COMPOUND (V) (mean diameter of microcapsules: 50pm) Chocolate Brown dyed. 15 Treatment C: Ti-Pure® R-931 + COMPOUND (V) (mean diameter of microcapsules: 25pm) undyed Treatment E: Ti-Pure® R-931 (mean diameter of microcapsules: 50pm) undyed. The following materials and methods were used during the 20 experiments described below. Compounds (III)-(V) were supplied by IACR-Rothamsted. Porcine gelatine (Type A, Isoelectric point 8) omniTechnik Microverkapselungs-Gmbh (Germany), Ethocel® 100 25 .(Ethylcellulose, a Dow Chemical Company product) Univar (Croydon), Eudragit® (E100) Degussa (Germany), Exxsol® (D 100) and Solvesso® (100) ExxonMobil (Belgium), Desmondur VL (Diphenylmethane-diisocyanate, MDI) Bayer (Germany), Ti-Pure® R-931. (Titanium dioxide) DuPont (Belgium) and Chocolate Brown 30 HT (Brown 3, CI 20285, E155) WS Simpson (London) were obtained as gifts. All other dyes and chemicals were purchased from Sigma-Aldrich chemical company (Dorset). Laboratory sprayer (Ecospray, Labo-Chemie-France) was purchased from Rotec WO 2006/077394 PCT/GB2006/000157 24 Scientific Limited (Milton Keynes). The results of the bioassays were analysed using Genstat 5th edition, release 4.2. Ultraviolet spectroscopy. 5 Ultraviolet (UV) absorbance spectra were recorded on a dual beam spectrophotometer (Shimadzu, UV-160A) using matched pair of quartz cuvettes of 1 cm path length. Spectra of all water soluble dyes were obtained as aqueous solutions in double distilled water. Spectra of all non water-soluble compounds 10 were obtained as solution in appropriate solvent. Typically spectra of dyes were recorded over 800-200 nm range. High performance liquid chromatography (HPLC). The HPLC system was from Waters comprising of two 510 pumps, a 15 717 plus Autosampler, a System Interface Module, a Lambda-Max 480 detector and Millennium Chromatography Manager software. Chromatography was achieved on a Zorbax ODS 5pm C18 analytical column of dimension 4.6 x 250 mm internal diameter maintained at 350 C. Mobile phases were unmodified water (Milli-Q grade) 20 in reservoir A and unmodified acetonitrile in reservoir B. Linear gradient elution was used with 70 to 90% B over the first 10 minutes, then 100% B for 3 minutes and returning to 70% B over 4 minutes. Injection cycle time was 20 minutes with a flow rate of 2 ml/min. The samples were either dissolved in 25 acetonitrile or diethyl ether and 10 pl volume injected on to the column which was maintained at '35 0 C. Prior to use, mobile phase were degassed under vacuum with sonication and continuously sparged with helium. The naphthoquinones were detected by measurement of UV absorbance at 269 nm. 30 Scanning electron microscopy. Microcapsule specimens were mounted on aluminium stubs and coated with gold in an Emscope SC500A sputter coater. Specimens 35 were examined and photographed with a Phillips XL20 scanning electron microscope.

WO 2006/077394 PCT/GB2006/000157 25 Identification of dyes suitable for photostabilisation of COMPOUND (V). Dyes with absorption spectra similar to Bismarck Brown R were 5 selected as potential candidates for dying microcapsules since Bismarck Brown was found to absorb UV light. Aqueous solutions of the dyes were prepared, an aliquot of each solution was transferred to a quartz cuvette and the UV absorbance spectrum recorded. The cuvette containing the solution was then 10 irradiated with 254 nm UV light, with the clear surface of the cuvette facing the radiation source, for various time periods and the spectra recorded again. Preparation of microspheres. 15 Microspheres containing ethylcellulose, Eurdagito E 100 or a mixture of ethylcellulose and Eudragite E 100 (3:1) were made by emulsifying a solution of the polymer mixture into an aqueous solution of gelatine. 20 Typically, 1 g of polymer or polymer mixture was dissolved in 40 ml of dichloromethane at room temperature. The polymer solution was dispersed in 130 ml of 2% (w/v) aqueous gelatine solution at 30'C with an Ultra Turrax@ homogeniser to give about 20 ptm droplets and the agitation continued through out 25 the rest of the procedure. The dispersion was warmed in a water bath to 40 0 C and maintained at that temperature for four hours. The system was then allowed to cool to room temperature, the resultant microspheres washed thoroughly with water and resuspended in 3 ml of water. The polymeric microspheres had a 30 mean size of about 5 pm diameter. Eurdragito E 100 polymer was leached from the ethylcellulose/Eudragito E 100 microspheres, by suspending them in 1M hydrochloric acid to provide porous ethylcellulose microspheres. 35 WO 2006/077394 PCT/GB2006/000157 26 Preparation of gelatine microcapsules. Gelatine microcapsules were produced by the complex coacervation method. Typically, the pH of 140 ml of 1.33% (w/v) aqueous gelatine (type A with isoelectric point 8) solution, 5 maintained at 45 0 C, was adjusted to 6.25 with 10% (w/v) sodium hydroxide. 15 ml of dibutylsebecate containing 1% by volume Span 85 , pre warmed to 45'C, was added to the gelatine solution and dispersed with a mechanical stirrer. The droplet size of the dibutylsebecate dispersion was adjusted and the 10 agitation continued through out the rest of the procedure. 3 ml of a 70% by weight aqueous dispersion of Ti-Puree R-931 was added to the dibutysebecate dispersion, followed by drop wise addition, over 10 minute period, of 30 ml of 0.5% by weight aqueous carrageenan (Type 1) solution at 45'C. The system was 15 then allowed to cool to room temperature slowly. Once the system had reached room temperature, it was chilled to 4 0 C using an ice bath and maintained at that temperature for one hour. 5 ml of 25% by weight aqueous gluteraldehyde solution was added to the chilled system and maintained for a further one 20 hour at 4 0 C. The ice bath was then removed, the system allowed to warm up and maintained at room temperature for about 18 hours. Naphthoquinone compounds (iii)-(V), when present, were 25 encapsulated as a solution in 15 ml of either ExxsolG D 100 or dibutylsebecate. Typically the microcapsules had a mean size of either 25 pm or 50 pm diameter. Microencapsulation was also carried out, in the presence of 30 each type of microspheres as per Ti-Puree R-931, to incorporate the particulate matter into the wall of the capsules to make them permeable. The capsules were harvested either as a slurry or wet cake. The microcapsules contained Span 85* (sorbitan trioleate) as a surfactant, to promote the translocation of WO 2006/077394 PCT/GB2006/000157 27 COMPOUND (V) into whitefly. Appropriate placebo microcapsules were produced to carry out preliminary tests and to act as controls in bioassay. 5 The presence of nano particles of Ti-Pure® R-931 inhibits aggregation of the dispersed droplets during production of the gelatine microcapsules. Sometimes during the preparation of the microcapsules, especially capsules below 50 microns, the dispersed droplets are encapsulated as aggregates resulting in 10 bigger capsules. Ti-Pure® R-931 inhibits this aggregation enabling discrete microcapsules of below 10 microns to be formed. These smaller microcapsules are easier to apply to a plant or an animal by spraying, as they do not clog up the nozzle of any spraying device. 15 Preparation of polyurethane microcapsules. Polyurethane microcapsules of COMPOUND (V), as a solution in SolvessoA 100, were produced by the interfacial polymerisation method using Desmondur VL and ethyleneglycol in the organic and 20 aqueous phase respectively. Typically, 15 ml of a 6.7% by volume solution of Desmondur VL in Solvesso@ 200 was dispersed in 120 ml of 5% (w/v) solution of gum acacia at room temperature. The droplet size of the dispersion was adjusted and the agitation continued through out the rest of the 25 procedure. 5 ml of ethyleneglycol was added dropwise to the dispersion and the system was 'warmed in a water bath to 60'C and maintained at that temperature for 18 hours..The system was then allowed to cool to room temperature and the resultant microcapsule slurry was diluted with water as required. 30 Typically the microcapsules had a size range of 5 to 30 pm in diameter. Encapsulation was also carried out in the presence of Chocolate Brown dissolved in the aqueous phase. Appropriate placebo microcapsules were produced to act as controls. 35 WO 2006/077394 PCT/GB2006/000157 28 Photo stabilisation study using Chocolate Brown dye. A batch of gelatine microcapsules containing 300 mg of COMPOUND (V) in 15 ml of Exxsol@ D100 was produced. The capsule slurry was washed repeatedly with water to remove 5 debris and filtered to obtain a wet cake. An aliquot of the wet cake (11.3 g) was made up to 50 ml and dyed brown with Chocolate Brown (500 mg, equivalent to 10 mg/ml solution). Aliquots (200 pl) of microcapsule slurry of brown and undyed capsules were applied to glass microscope slides in duplicate. 10 The slurry from each batch was spread to form a monolayer of microcapsules on each slide. The slides were air dried in a dark at room temperature (~21' C) and exposed to daylight on a south-facing windowsill for various time periods. Two slides from each batch were analysed per time point post of exposure. 15 Unexposed slides stored in the dark were used as time zero reference. The contents of the capsules were extracted by rupturing the capsules, by rolling a glass rod on the slides, and washing 20 both the rod and the slide with diethyl ether. The extracts were made up to 10 ml and assayed by HPLC. Examination of the slides under the microscope showed that the capsules were all broken and had released their contents. 25 Bioassay. Tomato plants used in the bioassays were grown in controlled glasshouse cubicles at 20' C, 12h Light: 12h Dark (12L: 12D) light regime, using 400 watt holophane daylight bulbs, to the third true leaf stage (approximately five weeks old from 30 sowing). Whiteflies were cultured on poinsettia (Euphorbia pulcherrima) maintained at 220 C, 16L: 8D light regime and 65% relative humidity. Adults were removed from stock culture when required for infestation of test plants. Bioassays were carried out "blind", i.e. all treatments were unknown to the 35 investigators throughout the trial. Phytotoxic effects, such as WO 2006/077394 PCT/GB2006/000157 29 scorching, leaf distortion necrosis or necrotic lesions were assessed at one week and one month intervals. Any signs were noted at each assessment period and photographs were taken of each treatment set. Any plant showing signs of phytotoxicity 5 was photographed. Determination of the optimum capsule density in the spray solution and preliminary phytotoxicity studies. Aliquots of Chocolate Brown dyed placebo gelatine microcapsule 10 (50 pm mean diameter) wet cake were made up to 50 ml with water to obtain 1/8, 1/4 and 1/2 dilution of capsules in a laboratory sprayer (Ecospray). Filter paper and both surfaces of tomato leaves were sprayed with the diluted capsule slurry. 15 The sprayed objects were allowed to air dry and the distribution of the capsules monitored both by naked eye and under a microscope. Representative areas (2 cm 2 ) were cut from the filter paper sprayed with 1/8 and 1/4 dilution of capsule slurry, sandwiched between two glass slides and viewed under 20 the microscope. The number of capsules present in the field of view (2.27 mm 2 ), at randomly selected areas of the filter paper, were counted. The sprayed tomato leaves were examined qualitatively under the microscope. 25 Either the top or the abaxial leaf surface of tomato plants was sprayed with either 1/8 or 1/4 dilution of capsule slurry in duplicate. Two plants were sprayed on both surfaces with 1/4 dilution of capsule slurry. All plants were transferred to the glasshouse and monitored for phytotoxic effects. 30 First Bioassay The First Bioassay was carried out according to the schematic protocol shown in Figure 1. The formulations used can be summarised as follows: 35 WO 2006/077394 PCT/GB2006/000157 30 Formulation Material Active A Gelatine/ethocel -dyed Compound V B Gelatine/ethocel -undyed Compound V C Polyurethane - undyed Compound V D emulsion Compound V E Gelatine/ethocel -dyed placebo F polyurethane - dyed Compound V Two absolute control tests (G, H) with no treatment were also carried out in daylight and subdued light respectively. 5 COMPOUND (V) microcapsule formulations contained 300 mg of the compound dissolved in 15 ml of solvent. Exxsolve® D 100 and Solvesso* 100 were used as solvents in gelatine and polyurethane microencapsulation procedures respectively. The microcapsule slurries were diluted to give 1000 ppm of COMPOUND 10 (V) with 1/6 dilution of capsules, in the final spray solutions. Aqueous Chocolate Brown solution (10 mg/ml) was used to produce dyed COMPOUND (V) formulations (A, E & F). None of the microcapsule formulations contained any surfactant in the capsules or in the aqueous dispersion medium. COMPOUND (V) 15 emulsion concentrate, supplied by Rothamsted, was diluted with water to produce formulation D having 1000 ppm of active ingredient. The abaxial surface of leaves of 18 tomato plants per 20 formulation were sprayed with the various formulations and allowed to dry in the dark. Six plants per formulation were maintained at 210 C (± 20 C), 16L: 8D artificial light (400 watt holophane day light bulb, supplementary lighting to come on 5am to 9pm; lighting on when ambient level drops below 25 100W/m 2 , off above 600W/m 2 , screens set to close at > 800W/m 2 ) regime with concomitant exposure to daylight in a glasshouse cubicle for 2 days ("day light"). Six plants per formulation were maintained at 220 C, 65% relative humidity, 16L: 8D regime WO 2006/077394 PCT/GB2006/000157 31 at 60 pmol/m 2 /sec light output ("subdued light") in a controlled environment (quarantine) room for 2 days. Following the 2-day "day light" exposure period, the plants 5 were transferred to the controlled environment room. All the plants in the controlled environment room were infested with whiteflies as per the protocol in Figure 1. Typically, adult whiteflies were collected in Petri dishes from the main stock culture, immobilised briefly with carbon dioxide and 10 transferred to clip cages after initial recovery. Photographs of an idealised clip cage arrangement on tomato plant leaves are shown in Figure 2a and 2b. Mortality (no movement of whitefly observed following mechanical stimulation) 15 rate of whiteflies in the clip cages were monitored over a seven-day period at 1, 2, 4 and 7 days post infestation. The remaining six plants per formulation were maintained in "day light" in a glasshouse cubicle for one month (phytotoxicity test). 20 Six untreated plants (absolute control) were maintained in "subdued light" in the controlled environment room for one month. Six untreated plants (absolute control) were maintained in a glasshouse cubicle for one month. All plants were 25 monitored for phytotoxicity. Second Bioassay The Second Bioassay was carried out according to the schematic protocol shown in Figure 3. Only gelatine microcapsule 30 formulations with Ti-Pure® R-931 incorporated in the capsule wall were used in the second bioassay. In this case the formulations used can be summarised as follows: 35 WO 2006/077394 PCT/GB2006/000157 32 Formulation Material Active Dyed/undyed incorporated into gelatine A Ti-Pure® R-931 Compound V undyed (50pm) * B Ti-Pure® R-931 Compound V dyed (50pm) C Ti-Pure R-931 Compound V undyed (25 pm) D Ti-Pure® R-931 Compound V dyed (25 pm) E Ti-Pure® R-931 control(25pm) undyed F No treatment N/A absolute control G Ti-Pure® R-931 control(25pm) dyed * Figures in parenthesis refer to mean microcapsule diameter in the formulation 5 COMPOUND (V) microcapsule formulations were produced with 300 mg of the compound dissolved in 15 ml of dibutylsebecate containing 1% (v/v) Span® 85. The microcapsule slurries were diluted to give 1000 ppm of COMPOUND (V) in 1 /6th dilution of capsules, in the final spray solutions. The final spray 10 solutions also contained 0.33% (v/v) Tween® 20 [POE (20) sorbitan monolaurate] as surfactant in the aqueous medium. The microcapsules in formulations B, D and G were dyed with 6.6 mg/ml solution of Chocolate Brown. 15 Formulations A and B had a mean capsule size of 50 pm diameter and all of the others were 25 pm. The abaxial surface of the leaves of 6 tomato plants per formulation were sprayed with the various formulations, allowed WO 2006/077394 PCT/GB2006/000157 33 to dry in the dark and transferred to the controlled environment room (see first bioassay). Six untreated plants were used as absolute control (F). It was noticed that the plants sprayed with undyed microcapsules showed phytotoxic 5 effects and these were eliminated from the bioassay. All remaining plants in the controlled environment room were infested with whiteflies as per the protocol in Figure 3. Mortality rate of whiteflies in the clip cages were monitored 10 over a seven-day period at 1, 2, 4 and 7 days post infestation. Results and Discussion Dyes which have similar UV absorption spectra to that of Bismarck Brown are given in Table 1 below. 15 Table 1. UV protection dyes for 1,4-naphthoquinone pesticides WATER NAME - nax SOLUBILITY REMARKS (mg/ml) Acid Orange 51 446 (water) 30 Sulphonic acid derivative, Acid Orange 63 424 (water) 50 Sulphonic acid derivative Acid Orange 74 455 (water) 20 Sulphonic acid derivative Bismark Brown R 468 (50% 70 Diazo Ethanol) Bismark Brown Y 457 (50% 50 Diazo Ethanol + HCl) Bromocresol 423 (Methanol) 6 Sulphonephthal Green ein Chlorophenol Red 575 (H20) 60 Sulphonephthal ein pH indicator WO 2006/077394 PCT/GB2006/000157 34 WATER NAME 2max SOLUBILITY REMARKS (mg/ml) Chrysoidin 449 (H20) 20 Monoazo pH indicator Congo Red 497(H20 + NaOH) 40 Diazo pH indicator m-Cresol Purple 436 (H20) 2 pH indicator Crocein Orange G 482 (H20) 40 Monoazo Darrow Red 502 (50% 1 Oxazine Ethanol) Direct Black 22 481 (H20) ? Polyazo Ethyl Orange 474 (H20) 100 Monoazo pH Ethyl Red 447 ( 0.lN 3 Monoazo pH NaOH) Methyl Red 493(Methanol + 2 Monoazo pH HCl) Mordant Brown 1 373/487 (H20) 60 Diazo Mordant Brown 4 500/374 60 Monoazo (hot (Ethanol) water) Mordant Brown 33 442 (H20) 20 Monoazo Mordant Brown 48 492 (H20) 40 Monoazo Chocolate Brown 459 (H20) 40 Diazo. Sulphonic acid derivative (Food dye) The chemical structure of Bismarck Brown is:- ' NH

H

2 N H2N N=N N=N N4 2HCI H-3C CH3 CH

CH

3 Bismarck Brown R WO 2006/077394 PCT/GB2006/000157 35 Bromcresol Green, Ethyl Orange, Ethyl Red, Mordant Brown 33, Mordant Brown 48, and Chocolate Brown were selected as candidates for dying microcapsules since these are environmentally acceptable dyes and are therefore preferable to 5 Bismark Brown, which is not environmentally acceptable. Chocolate Brown was found to have the best spectral characteristics and UV stability when exposed to 254 nm UV irradiation as shown in Figure 4 and 5. 10 Unlike Bismarck Brown, the reductive cleavage of azo bonds in Chocolate Brown does not result in the production of carcinogenic aromatic amines. This is the reason Chocolate Brown can be used as a food colorant. Therefore, Chocolate 15 Brown was selected for dying COMPOUND (V) microcapsules as a preferred dye. The -results of in vitro COMPOUND (V) photostabilisation studies carried out with undyed and Chocolate Brown dyed impervious 20 gelatine microcapsules are shown in Figure 6. The COMPOUND (V) calibration curve, used in this study, for quantitation of the compound by HPLC, is shown in Figure 7. Four standard solutions of COMPOUND (V) in acetonitrile, with three replicates per standard, were used to generate the calibration curve. 25 COMPOUND (V) in undyed capsules degraded progressively on continued exposure to daylight. The amount of COMPOUND (V) in these capsules was reduced to 60% of the initial amount after 6 hours exposure, 23% after 16 hours and only 6% after 40 hours 30 (equivalent to 8 hours daylight exposure over 5 days). In contrast, almost 80% of the initial amount of COMPOUND (V) was present in Chocolate Brown dyed capsules even after 88 hours (equivalent to 8 hours daylight exposure over 11 days) exposure to daylight. 35 WO 2006/077394 PCT/GB2006/000157 36 Since gelatine microcapsules are impervious to their contents, attempts were made to make them more pervious by incorporating particulate matter in the wall. To this end, ethylcellulose, and Eudragit E100 leached ethylcellulose microspheres were 5 made. The SEM micrographs of the various microspheres are shown in Figures 8a to 8d. The ethylcellulose microspheres have very small pores about 100 nm diameter(Fig. 8A). The acid washed 10 ethylcellulose/Eudragit& E1O microspheres (Ethylcellulose:Eudragito E100 [50:50]) have, in addition to the 100 nm diameter pores, a lot of large pores of about 2000 nm diameter(Fig. 8C). 15 The microspheres were incorporated into the gelatine microcapsule wall by carrying out the encapsulation in the presence of a specific type of microsphere dispersed in the aqueous phase. 20 The SEM micrographs of the microcapsules with the various types of microspheres embedded in the wall are shown in Figure 9a to 9b. Unlike gelatine, polyurethane did not take up Chocolate Brown 25. dye as effectively. Therefore, an alternative technique of incorporating the dye into the polyurethane wall was carried out. Microencapsulation was carried out with Chocolate Brown dissolved in the aqueous medium, so that the dye could be incorporated into the wall by the chemical reaction between the 30 isocyanate moieties in Desmondur VL and the hydroxy moieties in Chocolate Brown, at the oil/water interface: WO 2006/077394 PCT/GB2006/000157 37 OH Na3S.- -N=N <NN.Jt Sa ,OP N= ( 0, OH NaO 3 S N=N N=N SONa CHiOH Chocolate Brown HO O -NH- K CIt- CI1 2 .- \-NCO OCN-- \-CI-1 CH2- -NCO O0C -C t CH--- N~ - I C NCO Desmondur VL The microcapsules produced, were mostly aggregated and had faintly dyed walls surrounded by a brownish diffuse material. 5 Such particles could be used in formulations according to the present invention, since some dye was incorporated into the walls of the microcapsules. However, plain polyurethane microcapsules containing COMPOUND 10 (V) in Solvesso 100 (Desmondur VL does not disolve in Exxsol@ D100) were made, suspended in Chocolate Brown solution. Prior to carrying out the bioassays it was necessary to determine the appropriate capsule density in the spray solution 15 that would optimise the distribution of the capsules on the leaf surface. A microcapsule spray solution with 1/2 dilution of capsules was difficult to spray using the laboratory sprayer, Ecospray . The capsule distribution pattern obtained with 1/8 and 1/4 dilution of spray solution on filter paper is -20 shown in Figures 10a and 10b. Mean microcapsule distribution of about 1760 and 4490 capsules per cm2 were obtained with a 1/8 and 1/4 dilution of capsules respectively in the spray solution. Although, a superior 25 distribution of microcapsules was obtained with a 1/4 dilution, the high density of capsules in the spray solution tended to block the nozzle. Therefore, an intermediate dilution of 1/6 was chosen for carrying out the bioassay.

WO 2006/077394 PCT/GB2006/000157 38 The distribution of the microcapsules on both surfaces of tomato leaves was not as uniform as that obtained with filter paper. The capsules showed a tendency to accumulate around the vein area, predominantly in small aggregates as shown in Figure 5 10 c. Based on these studies, typically, COMPOUND (V) microcapsule slurry containing 300 mg of the compound was diluted to 300 ml to obtain 1/6 dilution of capsules having 1000 ppm of active 10 ingredient in the final spray solution. In preliminary toxicity evaluation, all tomato plants sprayed with Chocolate Brown dyed placebo gelatine microcapsule, either at 1/4 or 1/8 dilution of capsules, on both surfaces of leaf, 15 showed no phytotoxic effects. None of the treated plants in the first bioassay showed any phytotoxic effects during the whole assessment period. 20 Photographs of each treatment set at the end of each assessment period is shown in Figures Ila to 111. A low infestation of Western Flower Thrips (Frankliniella occidentalis) within the glasshouse test cubicle caused minor damage to some plants (which appear as white spots on leaves), but there were no 25 other visible signs. Normal growth was observed throughout the one-month assessment period. The results of efficacy evaluation of the various formulations of Compound (V) against Bemisia tabaci in the first bioassay 30 are given in Figure 11a to f. The weather over the day light exposure period was overcast. Mortality of flies in the absolute control (treatment G/H,) remained below 15% for the first four days of assessments, 35 increasing to 16.5% by day seven. In positive control (treatment D, Rothamsted emulsion formulation), the mortality WO 2006/077394 PCT/GB2006/000157 39 on the first day was 71% and 100% for plants exposed to daylight and subdued light respectively, which is indicative of minor degradation of the compound under daylight conditions. However, the mortality rate rose to 95% by day seven in 5 daylight exposed positive control. Mortality was slightly higher in all encapsulated formulations containing COMPOUND (V) (A, B, C, & F), which were maintained under subdued light. This result cannot be fully attributed to 10 the compound degrading in daylight conditions alone, because the Chocolate Brown dyed formulation A (gelatine/ethylcellulose) should provide photostability to the compound and shows equivalent potency to that of its counterparts in subdued light. 15 With formulation B (undyed gelatine/ethylcellulose), there was a large differance in mortality of flies between plants exposed to daylight and those maintained in subdued light. The overall mortality under subdued light conditions for B was comparable 20 to D, which implies that there was no problem with the release of the compound from B under subdued light conditions. If this was the case, under subdued light, A should have given equivalent results to that of B. 25 This difference may have been due to Chocolate Brown retarding the release of the compound. There is further evidence to support this hypothesis in that under subdued lighting, C (undyed polyurethane) gave a greater mortality than F (dyed polyurethane). 30 Another plausible explanation for the poor performance of the encapsulated formulations exposed to daylight appears to be the volatility of the solvents used, Exxsola D100 (distillation range, 235-270) and Solvessoa 100 (distillation range 163-180* 35 C). This appears to be the case for formulation C WO 2006/077394 PCT/GB2006/000157 40 (polyurethane), where the mortality of flies was poor in plants exposed to daylight. The organic phase in which COMPOUND (V) was dissolved did not contain any surfactants nor did the aqueous spray medium, which may have further contributed to the 5 overall poor performance. Mortality of flies increased with time in all microencapsulated formulations, which is indicative of slow release of the compound. Results indicate that ethylcelluose microspheres can 10 increase the permeability of the gelatine wall of the microcapsules. The results obtained in this bioassay prompted the reformulation of the compound and evaluation by the second 15 bioassay. The results of the efficacy evaluation of reformulated COMPOUND (V) against B. tabaci in the second bioassay are given in Figures 12a to 12e. Mortality of flies in the absolute control 20 (F) remained below 10% over the seven-day monitoring period. Although the mortality (34%) with the placebo formulation (G) was significantly higher than the absolute control, it was not much lower than the mortality (>90%) with formulations B & D. The only difference between B and D was the microcapsule 25 size,50 pm and 25 pm. The smaller capsule size (25 pm) was used to increase both the volume to surface area and capsule density on the leaf surface. Results showed that no significant improvement was achieved by reducing the capsule size. 30 These formulations contained fine particles of titanium dioxide, Ti-Pure® R-931, incorporated into the wall of the gelatine microcapsules to make them permeable. Two other types of titanium dioxide, Ti-Pure® R-902 and R-960, were also evaluated and found to be incompatible with gelatine solution.

WO 2006/077394 PCT/GB2006/000157 41 Ti-Pure® R-931 has 10.2% amorphous silica coating on the surface, which has an oil absorption capacity of 35.9 It appears that this coating of silica acts as a wick in 5 transferring the contents of the capsule to the flies on contact. These formulations contained Span* 85 in the organic phase within the capsules and Tween® 20 in the aqueous spraying medium to aid translocation of the active substance to the target and to aid in the wetting and spreading of the 10 formulation on the leaf surface respectively. Dibutylsebecate (DBS, boiling point: 178-179' C/3 mm Hg) was used as the solvent for COMPOUND (V). SEM micrographs of Ti-Pure® R-931 containing gelatine microcapsule are shown in Fig. 13a and 13b. It is evident from the micrograph of fractured capsule that the 15 particles traverse the wall Formulations containing undyed Ti-Pure® R-931 capsules (A, C & E) were found to be highly phytotoxic to tomato plants and were eliminated from the bioassay. 20 Photographs of phytotoxic effects on tomato plants are shown in Figures 14 and 15. Dying the capsules with Chocolate Brown, however, minimised the toxic effect. The capsules employed in the study had the maximum possible loading of Ti-Pure® 25 particles achievable under the microencapsulation conditions used. This was done to maximise the permeability of the capsules to demonstrate the desired effect on the target. Since it has been demonstrated here that Ti-Pure® makes the capsules permeable, it is anticipated that the phytotoxic effects could 30 be eliminated by reducing the loading of Ti-Pure® in the capsules with concomitant dying with Chocolate Brown. The use of titanium dioxide in microspheres to provide UV protection for bio pesticides (nuclear polyhedrosis virus, 35 which is a stomach poison) has been reported by Bull, D.L.

WO 2006/077394 PCT/GB2006/000157 42 (Formulations of microbial insecticides: microencapsulation and adjuvants. Formulation and application of microbial insectcides. A symposium at the Annual Meeting of the Entamological Society of America-Honolulu, Hawaii; December 1, 5 1976, Ed. Ignoffo, C.M.; Falcon, L.A., Miscellaneous Publications of tthe Entamological Society of America, Vol. 10, p 11-20(1978)). These water-insoluble, but digestible, microsphere formulations were made by a spray-drying, phase separation process. These workers, however, did not report the 10 type of titanium dioxide used or any phytotoxic effects. Two possible mechanisms may be responsible for the observed phytotoxicity. Ti-Pure® R-931 is coated with a high amount of amorphous silica, which may act by desiccating the leaf tissue. Plants exhibiting phytotoxicity appear to be more susceptible 15 to water stress than the others. Secondly, titanium dioxide is a photo catalyst, which chemically decomposes water molecules into highly reactive hydroxyl ions (OH-) under the influence of UV irradiation. 20 Conclusions Several dyes have been identified in this study as environmentally acceptable for photostabilising pesticides within microcapsule formulations. In particular the reductive cleavage of azo bonds in Chocolate Brown does not result in the 25 production of carcinogenic aromatic amines and as such it is permitted for use as a food colorant. COMPOUND (V) was stabilised within impervious gelatine microcapsules for periods in excess of eleven-days exposure to daylight. 30 Success in producing permeable microcapsules was achieved by incorporating ethylcellulose microspheres into the gelatin microcapsule wall. Similar results were also obtained with polyurethane microcapsule formulations. The incorporation of Ti-Pure®R-931 (titanium dioxide) produced capsules with further 35 improved performance, resulting in more than 95% mortality of 43 whiteflies Ti-Purea. R-931 incorporated microcapsules were found to be highly phytotoxic to tomato plants. However, dying these capsules with Chocolate Brown reduces the phytotoxic effects of Ti-Puree R 931 considerably. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise",. and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. The use of a dye for the protection of an active component contained within a microcapsule from U.V. 5 degradation, wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, 10 Mordant Brown 33, Mordant Brown 48 or Chocolate Brown.

2. The use according to claim 1 wherein the dye is incorporated within or located on the surface of the microcapsule. 15

3. The use according to claim 1 or 2 wherein the microcapsule comprises gelatine or polyurethane.

4. The use according to any one of claims 1 to 3 wherein the 20 active component is a pharmaceutical, cosmetic or agrochemical.

5. The use according to claim 4 wherein the active component is an agrochemical, and the agrochemical is a pesticide. 25

6. The use according to claim 5 wherein the pesticide is a naphthoquinone derivative of formula (I) 0 R R 2 0 (1) where R' is selected from an optionally substituted alkyl group, a hydroxy group or a group -OCOR 4 where R 4 is selected 45 from hydrogen, C 1 1 2 alkyl, C 1 1 2 haloalkyl, Ci 1 . 2 hydroxyalkyl, Ci 1 2 carboxyalkyl, phenyl or benzyl.

7. The use according to any one of claims 1 to 6 wherein the 5 microcapsule has an average diameter of less than 60pm.

8. The use according to any one of claims 1 to 7 wherein the microcapsule comprises a permeable wall. 10

9. The use according to claim 8 wherein the microcapsule has a particulate matter located in a wall thereof to render the wall permeable, the particulate matter comprises particles of a metal or an insoluble salt thereof or an insoluble polymeric material, and at least some of the particulate matter is coated 15 with silica.

10. The use according to claim 9 wherein the particulate matter is combined with a leachable material. 20

11. A formulation comprising an agrochemical, a pharmaceutical or a cosmetic, a microcapsule and a dye, wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, 25 Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown.

12. A formulation according to claim 11 wherein the dye is 30 incorporated within or located on the surface of the microcapsule.

13. A formulation according to claim 11 or 12 wherein the microcapsule comprises gelatine or polyurethane. 35 46

14. A formulation according to any one of claims 11 to 13 wherein the active component is a pharmaceutical, cosmetic or agrochemical. 5

15. A formulation according to claim 14 wherein the active component is an agrochemical, and the agrochemical is a pesticide.

16. A formulation according to claim 15 wherein the 10 agrochemical is a naphthoquinone derivative of formula (I) 0 R' R2 0 (I) where R 1 is selected from an optionally substituted alkyl group, a hydroxy group or a group -OCOR 4 where R 4 is selected from hydrogen, C 1 - 12 alkyl, C 1 - 12 haloalkyl, Ci- 12 hydroxyalkyl, 15 C 1 - 12 carboxyalkyl, phenyl or benzyl.

17. A formulation according to any one claims 11 to 16 wherein the microcapsule has an average diameter of less than 60 tm. 20

18. A formulation according to any of claims 11 to 17 wherein the microcapsule comprises a permeable wall.

19. A formulation according to claim 18 wherein the 25 microcapsule has a particulate matter located in a wall thereof to render the wall permeable, the particulate matter comprises particles of a metal or an insoluble salt thereof or an insoluble polymeric material, and at least some of the particulate matter is coated with silica. 47

20. A formulation according to claim 19 wherein the particulate matter is combined with a leachable material. 5

21. A microcapsule which contains an agrochemical and a dye, wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, 10 Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown.

22.A reagent delivery device containing a formulation according to any one of claims 11 to 20 or a microcapsule 15 according to claim 21.

23. A method for protecting a plant, said method comprising administering to the plant or its environment a formulation comprising (i) a microcapsule comprising an active component, 20 and (ii) a dye selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant 25 Brown 33, Mordant Brown 48 or Chocolate Brown, wherein components (i) and (ii) can be applied together or in separate stages, and wherein the active component is an agrochemical.

24. A method for producing an agriculturally effective 30 microcapsule comprising encapsulating an agrochemical within the microcapsule, wherein the surface of the microcapsule is dyed and/or dye is incorporated into the microcapsule during its preparation, wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark 35 Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct 48 Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown.

25. A method according to claim 23 or 24 wherein a silica 5 coated particle is positioned in the wall of the microcapsule to render said wall permeable.

26. The use of a dye, wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, 10 Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m-crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown, as a safener for the reduction of the 15 phytotoxic effects of reagents on plants.

27. A method for protecting an active ingredient encapsulated within a microcapsule from U.V. degradation comprising dyeing the surface of the microcapsule and/or incorporating dye into 20 the microcapsule and/or suspending the microcapsule in a dye wherein the dye is selected from Acid Orange 51, Acid Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, 25 Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown.

28. A formulation comprising an active component, a microcapsule and a dye selected from Acid Orange 51, Acid 30 Orange 63, Acid Orange 74, Bismark Brown R, Bismark Brown Y, Bromocresol Green, Chlorophenol Red, Chrysoidin, Congo Red, m crestol Purple, Crocein Orange G, Darrow Red, Direct Black 22, Ethyl Orange, Ethyl Red, Mordant Brown 1, Mordant Brown 4, Mordant Brown 33, Mordant Brown 48 or Chocolate Brown. 35 49

29. The use according to claim 1 or 26; a formulation according to claim 11 or 28; a microcapsule according to claim 21; a reagent delivery device according to claim 22; or a method according to any one of claims 23, 24 and 27; 5 substantially as hereinbefore described with reference to any one of the examples.